Challenges for the self-assembly of poly(Ethylene glycol)-poly(lactic acid) (PEG-PLA) into polymersomes : beyond the theoretical paradigms

We acknowledge support from the State of São Paulo Research Foundation (FAPESP-Brazil, projects 2013/08617-7 (Thematic project) and 2014/10456-4 (Apolinário, A.C. PhD fellowship) and the National Council for Scientific and Technological Development (CNPq- Brazil, project 303334/2014-2).Polymersomes (PL), vesicles formed by self-assembly of amphiphilic block copolymers, have been described as promising nanosystems for drug delivery, especially of biomolecules. The film hydration method (FH) is widely used for PL preparation, however, it often requires long hydration times and commonly results in broad size distribution. In this work, we describe the challenges of the self-assembly of poly (ethylene glycol)-poly(lactic acid) (PEG-PLA) into PL by FH exploring different hydrophilic volume fraction (f) values of this copolymer, stirring times, temperatures and post-FH steps in an attempt to reduce broad size distribution of the nanostructures. We demonstrate that, alongside f value, the methods employed for hydration and post-film steps influence the PEG-PLA self-assembly into PL. With initial FH, we found high PDI values (>0.4). However, post-hydration centrifugation significantly reduced PDI to 0.280. Moreover, extrusion at higher concentrations resulted in further improvement of the monodispersity of the samples and narrow size distribution. For PL prepared at concentration of 0.1% (m/v), extrusion resulted in the narrower size distributions corresponding to PDI values of 0.345, 0.144 and 0.081 for PEG45-PLA69, PEG114-PLA153 and PEG114-PLA180, respectively. Additionally, we demonstrated that copolymers with smaller f resulted in larger PL and, therefore, higher encapsulation efficiency (EE%) for proteins, since larger vesicles enclose larger aqueous volumes.Publisher PDFPeer reviewe

Solubilização micelar do ibuprofeno: influência do grupo polar dos tensoativos no grau de solubilização

An important property of micelles with particular significance in pharmacy is their ability to increase the solubility of poorly soluble drugs in water, thus increasing their bioavailability. In this work, the solubilization of ibuprofen (IBU) was studied in micellar solutions of three surfactants possessing the same hydrocarbon tail but different hydrophilic head groups, namely sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB), and n-dodecyl octa(ethylene oxide) (C12EO8). The results showed that, irrespective of the surfactant type, the solubility of IBU increased linearly with increasing surfactant concentration, as a consequence of the association between the drug and the micelles. The 80 mM DTAB and the 80 mM C12EO8 micellar solutions resulted in a 16-fold increase in solubility of IBU when compared to the buffer solution, whereas the 80 mM SDS micellar solution resulted in a 5.5-fold increase in IBU solubility. The highest value of molar solubilization capacity (chi) was obtained with DTAB, chi = 0.97, followed by C12EO8 ,chi = 0.72, and finally SDS, chi = 0.23. However, due to the stronger tendency of the nonionic surfactant in forming micelles in solution, at the same surfactant concentration, we obtained the same solubility of IBU in both DTAB and C12EO8.Uma propriedade importante das micelas, do ponto de vista farmacêutico, refere-se ao potencial destas em solubilizar fármacos pouco solúveis em água, aumentando sua biodisponibilidade. No presente trabalho, estudou-se a solubilização de ibuprofeno (IBU) em soluções micelares constituídas de três tensoativos apresentando a mesma cauda apolar, porém diferentes grupos hidrofílicos. Os tensoativos estudados foram dodecil sulfato de sódio (SDS), brometo de dodeciltrimetilamônio (DTAB) e óxido de n-dodecil octaetileno (C12EO8). De acordo com os resultados obtidos, a solubilidade do IBU aumentou linearmente com o aumento da concentração de todos os tensoativos estudados, devido às interações entre as micelas e o fármaco. O fármaco IBU apresentou um aumento de 16 vezes em sua solubilidade na presença de DTAB 80 mM e de C12EO8 80 mM. Por outro lado, na presença de SDS 80 mM a solubilidade do IBU aumentou apenas 5,5 vezes. O maior valor para o parâmetro capacidade molar de solubilização (chi) foi observado com o tensoativo DTAB, chi = 0,97, seguido pelo C12EO8, chi = 0,72 e, finalmente, o SDS, chi = 0,23. Entretanto, devido à grande tendência do C12EO8 em formar micelas, o perfil de solubilidade do IBU foi semelhante em DTAB e C12EO8

Polymeric micelles and molecular modeling applied to the development of radiopharmaceuticals

Micelles composed of amphiphilic copolymers linked to a radioactive element are used in nuclear medicine predominantly as a diagnostic application. A relevant advantage of polymeric micelles in aqueous solution is their resulting particle size, which can vary from 10 to 100 nm in diameter. In this review, polymeric micelles labeled with radioisotopes including technetium (99mTc) and indium (111In), and their clinical applications for several diagnostic techniques, such as single photon emission computed tomography (SPECT), gamma-scintigraphy, and nuclear magnetic resonance (NMR), were discussed. Also, micelle use primarily for the diagnosis of lymphatic ducts and sentinel lymph nodes received special attention. Notably, the employment of these diagnostic techniques can be considered a significant tool for functionally exploring body systems as well as investigating molecular pathways involved in the disease process. The use of molecular modeling methodologies and computer-aided drug design strategies can also yield valuable information for the rational design and development of novel radiopharmaceuticals.Micelas poliméricas compostas de copolímeros ligadas a um elemento radioativo são utilizadas em Medicina Nuclear com aplicação predominantemente diagnóstica. A vantagem relevante da utilização de micelas poliméricas em solução aquosa é o tamanho de suas partículas, as quais podem variar de 10 a 100 nm de diâmetro. Neste trabalho de revisão são apresentadas micelas poliméricas marcadas com radioisotopos, como tecnécio-99m (99mTc) e índio-111 (111In), assim como suas aplicações clínicas em técnicas de diagnóstico como Tomografia por emissão de Fóton Único (Single photon Emission Computed Tomography - SPECT), cintilografia, e Ressonância Magnética Nuclear (RMN). Neste contexto, sua aplicação em diagnóstico de sistema linfático e linfonodo sentinela recebe atenção especial. O emprego de técnicas de diagnóstico pode ser considerado uma ferramenta importante para a exploração de sistemas no organismo humano assim como para a investigação de caminhos moleculares envolvidos nos processos de diversas doenças. O uso de metodologias de modelagem molecular e estratégias de desenvolvimento de fármacos assistidas computacionalmente também pode fornecer informações valiosas para o planejamento e o desenvolvimento racional de novos radiofármacos

Protein PEGylation for the design of biobetters: from reaction to purification processes

The covalent attachment of polyethylene glycol (PEG) to therapeutical proteins is an important route to develop biobetters for biomedical, biotech and pharmaceutical industries. PEG conjugation can shield antigenic epitopes of the protein, reduce degradation by proteolytic enzymes, enhance long-term stability and maintain or even improve pharmacokinetic and pharmacodynamics characteristics of the protein drug. Nonetheless, correct information in terms of the PEGylation process from reaction to downstream processing is of paramount importance for the industrial application and processing scale-up. In this review we present and discuss the main steps in protein PEGylation, namely: PEGylation reaction, separation of the products and final characterization of structure and activity of the resulting species. These steps are not trivial tasks, reason why bioprocessing operations based on PEGylated proteins relies on the use of analytical tools according to the specific pharmaceutical conjugate that is being developed. Therefore, the appropriate selection of the technical and analytical methods may ensure success in implementing a feasible industrial process

Methods of endotoxin removal from biological preparations : a review

ABSTRACTPURPOSE: Endotoxins, also called lipopolysaccharides (LPS), are major contaminants found in commercially available proteins or biologically active substances, which often complicate study of the biological effects of the main ingredient. The presence of small amounts of endotoxin in recombinant protein preparations can cause side effects in host organism such as endotoxin shock, tissue injury, and even death. Due to these reactions, it is essential to remove endotoxins from drugs, injectables, and other biological and pharmaceutical products. An overview of this subject is provided by this article. METHODS: An extensive review of literature with regard to methods for removal of endotoxin from biotechnological preparations was carried out. RESULTS: A short history of endotoxin is presented first. This is followed by a review of chemical and physical properties of endotoxin and its pathophysiological effects when the body is exposed to LPS excessively or systemically. The techniques of endotoxin determination and interaction of endotoxin with proteins is also presented, taking into consideration the established techniques as well as the state of the art technology in this field. A review of techniques of endotoxin mentioned with relatively high protein recoveries; however, special attention is given to two-phase aqueous micellar systems, which are valuable tools for endotoxin removal from pharmaceutical proteins on a small scale because they provide a mild environment for biological materials. CONCLUSIONS: Efficient and cost-effective removal of endotoxins from pharmaceutical and biotechnology preparations is challenging. Despite development of novel methods, such as the two- phase aqueous micellar systems, in recent years, more research is needed in this field

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30-40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.publishe

Poly (lactic-co-glycolic acid) nanospheres allow for high l-asparaginase encapsulation yield and activity

L-asparaginase (ASNase) is an amidohydrolase used as a chemotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL). The nanoencapsulation of this enzyme is strategic to avoid its immediate immunogenic effects that lead to a decrease in the enzyme half-life. In this work, ASNase-containing nanoparticles (NPs) were prepared by double emulsification, through an ultrasonic sonicator or an ultra-turrax, using two copolymers of 50:50 (w/w) poly (lactic-co-glycolic acid) (PLGA) with different ranges of molecular weight (24-38 kDa and 30-60 kDa) and varying the concentration of polyvinyl alcohol (PVA) as a stabilizer (0.5, 1.0, 1.5 and 2.0%) as well as the emulsification time (30 and 60 s). Using 24-38 kDa PLGA and 1.0% PVA, we obtained by cavitation NPs with hydrodynamic diameter of 384 nm, polydispersity index of 0.143 and Zeta potential of -16.4 mV, whose ASNase encapsulation efficiency was as high as 86.67 \ub1 1.84%. The encapsulated enzyme showed an activity 22% higher than that of the free enzyme, and no conformational changes were detected by circular dichroism. The enzyme release from NPs entrapped in dialysis bags (500 kDa molecular weight cut-off) allowed selecting a controlled system able to release about 30% of the enzyme within 48 h, for which the Korsmeyer-Peppas model provided the best correlation (R2 = 0.9265)